Anti-cd40l antibodies and methods for treating cd40l-related diseases or disorders

ABSTRACT

Anti-human CD40L antibodies engineered to lack the ability to activate platelets and methods for treating patients having a CD40L-associated disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.16/125,317 filed on Sep. 7, 2018, which is a Divisional of U.S.application Ser. No. 15/667,477 filed on Aug. 2, 2017, which is aContinuation application of, and claims priority to, PCT InternationalApplication No. PCT/US2016/016165, filed Feb. 2, 2016, which claims thebenefit under 35 USC § 119 of U.S. Provisional Application No.62/111,261, filed Feb. 3, 2015, the entire contents of theaforementioned disclosures are hereby incorporated by reference.

FIELD

Anti-CD40L antibodies, compositions comprising the antibodies, andmethod of using same for treatment of CD40L-related diseases ordisorders.

SEQUENCE LISTING

This application contains a Sequence Listing which is submitted herewithin electronically readable format. The electronic Sequence Listing filewas created on Dec. 14, 2018, is named “224823-436510-Amended_ST25.txt”and has a size of 43 KB. The entire contents of the Sequence Listing inthe electronic “224823-436510-Amended_ST25.txt” file are incorporatedherein by this reference.

BACKGROUND

The interaction of CD40 with its ligand CD40L plays a critical role inregulating immune responses. Binding of CD40L to CD40 triggersactivation of the CD40 pathway which up-regulates costimulatorymolecules such as CD80 and CD86. Blockade of the interaction betweenCD40 and CD40L by monoclonal antibodies has been shown to result inprotection from autoimmunity and graft rejection in various preclinicalmodels. Recently, in a mouse model of amyotrophic lateral sclerosis, anantibody directed to CD40L was shown to delay disease onset and prolongsurvival the onset of disease. (U.S. Pat. No. 8,435,514, herebyincorporated by reference). In early clinical studies, the humanizedanti-CD40L antibody hu5c8 showed efficacy in patients with lupus and inpatients with immune thrombocytopenic purpura. However, incidents ofthromboembolism in the patients treated with hu5c8 halted furthertrials. Further in vitro and preclinical animal studies established thatinteraction of the Fc with the Fc receptor FcγRIIa caused plateletactivation, and aggregation, that resulted in thromboembolic events.Various approaches have been taken to reduce or eliminate theinteraction of the immunoglobulin Fc region with FcγRIIa, includingintroducing a point mutation in the Fc region to make an aglycosylatedanti-IC40L IgG1 which lacked Fc effector function. Other approaches usefragments of antibodies lacking the Fc region or antibodies that containmultiple amino acid substitutions in the Fc region. Although theanti-CD40L antibody, hu5c8, showed efficacy in human patients there isno anti-CD40L antibody on the market. Accordingly, there is a need forimproved anti-CD40L antibodies for administration to humans that do notcause platelet activation or aggregation yet are stable and bind toCD40L.

SUMMARY

The present invention provides anti-CD40L antibodies, suitable for usein humans and non-human primates, having an Fc domain that has beenengineered to reduce or eliminate platelet aggregation and theconcomitant risk of thromboembolism. In one aspect of the invention, thepresent invention provides antibodies that are humanized versions of themouse anti-human CD40L antibody 5c8. In one embodiment an antibody ofthe present invention comprises a human IgG1 consensus framework whereinthe variable light chain and the variable heavy chain comprise the CDRsequences of 5c8.

One aspect of the present invention is an isolated antibody that bindsto CD40L and that comprises a light chain and a heavy chain, wherein (i)the light chain comprises a light chain variable region comprising anamino acid sequence having at least 95% sequence identity with SEQ IDNO:1; (ii) the heavy chain comprises a heavy chain variable region andan Fc region wherein a) the heavy chain variable region comprises anamino acid sequence having at least 95% sequence identity with SEQ IDNO:2; and b) the Fc region comprises an amino acid sequence having atleast 95% sequence identity with SEQ ID NO:3 wherein the Fc regioncomprises one or a combination of substitutions selected from the groupconsisting of C11S, C14S, and P23S. Optionally the Fc region comprises afurther amino acid substitution C5S.

Another aspect of the present invention is a method for treating asubject with a CD40L-associated disease or disorder comprisingadministering to the subject a therapeutically effective amount of anantibody according to the invention. One embodiment of the presentinvention is a method for treating a subject with a neurodegenerative orneuromuscular disease or disorder; an inflammatory or immune disease ordisorder; or an autoimmune disease, comprising administering to thesubject a therapeutically effective amount of an antibody according tothe invention. Another embodiment is a method for treating a subjectwith a CD40L-associated disease or disorder comprising administering tothe subject a therapeutically effective amount of an antibody accordingto the invention administered in combination with a compound that blocksthe interaction between CD28 and CD86 or between CD28 and CD80.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C show the heavy chain amino acid sequences for hu5c8(FIG. 1A), JB5 (FIG. 1B) and JB5-K74R (FIG. 1C). The amino acids shownin bold type indicate amino acids that differ between the heavy chainsequences for 5c8 and the heavy chain sequences for JB5 and JB5-K74R.

FIGS. 2A-2D show the light chain amino acid sequence for JB5 (FIG. 2A),the light chain amino acid sequence for JB5-R28K (FIG. 2B), the Fcregion amino acid sequence for hu5c8 (FIG. 2C), and the Fc region aminoacid sequence for JB5 (FIG. 2D). The amino acids shown in bold typeindicate the amino acids that differ between the light chain sequencesfor 5c8 and JB5-R28K and between the Fc regions for hu5c8 and JB5.

FIG. 3 is a graph showing the relative binding to human CD40L, of JB5antibody (circles, dotted line), hu5c8 antibody (squares-solid line),and the control CTLA4-IgG1 (triangles).

FIG. 4 is a graph showing the binding of hu5c8 antibody to FCGR1A(circle, solid line) (SEQ ID NO:22), FCGR2A (circle, dotted line) (SEQID NO:23), FCR3A (SEQ ID NO:24) and FCR3B (SEQ ID NO:25) isoforms of thehuman Fc gamma receptor protein.

FIG. 5 is a graph showing that JB5 antibody to FCGR1A (SEQ ID NO:22),FCGR2A (SEQ ID NO:23), FCR3A (SEQ ID NO:24), or FCR3B (SEQ ID NO:25)isoforms of the human Fc gamma receptor protein.

FIG. 6 shows the analytical chromatography elution profile for JB5antibody run at 30° C. from a size exclusion column.

FIG. 7 shows the analytical chromatography elution profile for hu5c8antibody run at 30° C. from a size exclusion column.

FIG. 8 is a graph showing the binding of the platelet activation markerPAC1 antibody to untreated platelet samples (negative control), asassessed by fluorescence activated cell sorting (FACS).

FIG. 9 is a graph showing the binding, as assessed by FACS, of ananti-PAC1 antibody

FIG. 10 is a graph showing the binding, as assessed by FACS, of ananti-PAC1 antibody to platelets after the incubation of the plateletswith CD40L.

FIG. 11 is a graph showing the binding, as assessed by FACS, of ananti-PAC1 antibody to platelets after incubation of the platelets withan immune complex of CD40L and hu5c8 antibody.

FIG. 12 is a graph showing the binding, as assessed by FACS, of ananti-PAC1 antibody to platelets after incubation of the platelets withan immune complex of CD40L and JB5 antibody.

FIG. 13 is a graph showing the binding, as assessed by FACS, of ananti-PAC1 antibody to platelets after incubation of the platelets withan immune complex of CD40L and the hu5c8 F(ab′)₂.

FIG. 14 is a scatter plot graph showing FACS results from three persons'platelets after incubation of the platelets with 20 μM ADP, 5 μg/mlCD40L, the immune complex of CD40L and hu5c8, the immune complex ofCD40L and JB5 antibody or the immune complex of CD40L with hu5c8F(ab′)₂.

FIG. 15 provides the variable light region amino acid sequence of theanti-CD40L antibodies JB5 and hu5c8 (SEQ ID NO:1), the variable heavyregion amino acid sequence of the anti-CD40L antibodies JB5 and hu5c8(SEQ ID NO:2), the Fc region amino acid sequence of the anti-CD40Lantibody hu5c8 (SEQ ID NO:3), the Fc region amino acid sequence of theanti-CD40L antibody JB5 (SEQ ID NO:4), the variable light region aminoacid sequence of the anti-CD40L antibody JB5-R28K (SEQ ID NO:5), thevariable heavy region amino acid sequence of the anti-CD40L antibodyJB5-K74R (SEQ ID NO:6), and the light chain amino acid sequence of theanti-CD40L antibody JB5 (SEQ ID NO:7).

FIG. 16 provides the light chain synthetic nucleotide sequence thatencodes the anti-CD40L antibody JB5 (SEQ ID NO:8), upper case lettersrepresent the exons and the lower case letters represent the intronsequences of the synthetic gene, and also provides the heavy chain aminoacid sequence of the anti-CD40L antibody JB5 (SEQ ID NO:9).

FIG. 17 provides a synthetic nucleic acid sequence that encodes theheavy chain of the anti-CD40L antibody JB5 (SEQ ID NO:10), upper caseletters represent the exons and the lower case letters represent theintron sequences of the synthetic gene.

FIG. 18 provides the amino acid sequence of the anti-CD40L antibodyJB5-R28K (SEQ ID NO:11), a synthetic nucleic acid sequence that encodesthe light chain of the anti-CD40L antibody JB5-R28K (SEQ ID NO:12),upper case letters represent the exons and the lower case lettersrepresent the intron sequences of the synthetic gene, and also providesthe heavy chain amino acid sequence of the anti-CD40L antibody JB5-K74R(SEQ ID NO:13).

FIG. 19 provides a synthetic nucleic acid sequence that encodes theheavy chain of the anti-CD40L antibody JB5-K74R (SEQ ID NO:14) uppercase letters represent the exons and the lower case letters representthe intron sequences of the synthetic gene.

FIG. 20 provides the amino acid sequences of the CDRs of the heavy andlight chain of the anti-CD40L antibody JB5 (SEQ ID NOs:15-20,respectively) and the amino acid sequence of the hu5C8 heavy chain (SEQID NO: 21).

DETAILED DESCRIPTION Definitions

The terms such as “comprises”, “comprised”, “comprising”, “contains”,“containing” and the like have the meaning attributed in United Statespatent law; these terms are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in United States patent law; these termsallow for the inclusion of additional ingredients or steps that do notmaterially affect the basic and novel characteristics of the claiminvention. The terms “consists of” and “consisting of” have the meaningascribed to them in United States patent law; these terms are closeended.

The terms “treat,” “treatment” and the like, include therapeutictreatment and prophylactic treatment. Therapeutic treatment is treatmentof a subject that has signs or symptoms of the disease, condition ordisorder to be treated. Prophylactic treatments refers to treatment of asubject that is predisposed to the disease, condition or disorder thatdoes not show overt signs of the disease, condition or disorder. Thus,treatment may result in stasis of, partial or total alleviation, orreduction of signs or symptoms of illness, and specifically includes,without limitation, prolongation of survival.

About” indicates that the stated numerical value allows some slightimprecision (with some approach to exactness in the value; approximatelyor reasonably close to the value; nearly). If the imprecision providedby “about” is not otherwise understood in the art with this ordinarymeaning, then “about” as used herein indicates at least variations thatmay arise from ordinary methods of measuring and using such parameters.In addition, disclosure of ranges includes disclosure of all values andfurther divided ranges within the entire range.

The use of the conjunction “or” is used interchangeably with at “leastone of”. For example: where a composition comprises A or B, the methodmust comprise at least one of A and B but may also comprise both A andB. Likewise a composition comprising “A, B, C or D” must comprise atleast one of the group of A, B, C and D, but may also comprise all orany combination of A, B, C and D.

Amino acid substitutions are denoted by the convention in which theoriginal amino acid, the position of the amino acid in the specifiedsequence and the replacement amino acid are identified, for example,C11S would indicate that the cysteine at position 11 of the polypeptidesequence is replaced with a serine.

“5c8” refers to the mouse anti-human antibody that binds CD40L and isproduced by the hybridoma that is available from the ATCC having theaccession number HB10916 and is described in U.S. Pat. No. 5,474,771.“hu5c8” refers to a humanized version of 5c8 the sequence of which isdisclosed in Karpusas, et al., Structure vol. 9, pp 321-329, (2001).

Reference in the specification is made to percent identity betweenpolypeptide or amino acid sequences. The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. Identity can be measured as “local identity” or “globalidentity”. Local identity refers the degree of sequence relatednessbetween polypeptides as determined by the match between strings of suchsequences. Global identity refers to the degree of sequence relatednessof a polypeptide compared to the full-length of a reference polypeptide.Unless specified otherwise, as used herein, identity means globalidentity. For the purposes of this disclosure, the percentages forglobal identity are calculated using Needleman and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm using a Blossum 62 scoring matrix witha gap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5. There are many publically available software programs thatincorporate the Needleman and Wunsch algorithm, e.g. the GAP program inthe GCG software package.

CD40L is also known as CD154, gp39, T-BAM, 5c8 antigen, or TNF relatedactivation protein (TRAP).

EMBODIMENTS

The present invention provides for therapeutic anti-human CD40Lantibodies and methods for using the antibodies of the invention fortreating patients with a CD40L-associated disease or disorder. Variousexemplary embodiments of the present invention are provided, however,the invention is to be limited by the claims and not the disclosedembodiments.

In one aspect of the invention, the present invention providesantibodies that are modified versions of the anti-CD40L antibody hu5c8that comprise a human IgG1 consensus framework having the variable lightchain and the variable heavy chain CDR sequences of hu5c8 with an Fcdomain modified to prevent platelet activation.

Table 1 provides a description of the SEQ ID NOs referenced in theapplication.

TABLE 1 SEQ ID NO: Description of Sequence 1 Light chain variable regionamino acid sequence (hu5c8 and JB5) 2 Heavy chain variable region aminoacid sequence (hu5c8 and JB5) 3 Fc region amino acid sequence (hu5c8) 4JB5 Fc region amino acid sequence 5 JB5-R28K light chain variable regionamino acid sequence 6 JB5-K74R heavy chain variable region amino acidsequence 7 JB5 light chain amino acid sequence 8 JB5 light chain nucleicacid sequence 9 JB5 heavy chain amino acid sequence 10 JB5 heavy chainnucleic acid sequence 11 JB5-R28K light chain amino acid sequence 12JB5-R28K light chain synthetic gene nucleic acid sequence 13 JB5-K74Rheavy chain amino acid sequence 14 JB5-K74R heavy chain synthetic genenucleic acid sequence 15 CDR-1 of the JB5 Variable Light Chain aminoacid sequence 16 CDR-2 of the JB5 Variable Light Chain amino acidsequence 17 CDR-3 of the JB5 Variable Light Chain amino acid sequence 18CDR-1 of the JB5 Variable Heavy Chain amino acid sequence 19 CDR-2 ofthe JB5 Variable Heavy Chain amino acid sequence 20 CDR-3 of the JB5Variable Heavy Chain amino acid sequence 21 Hu5c8 Heavy Chain amino acidsequence

One embodiment (embodiment A) is an isolated antibody that binds toCD40L and that comprises a light chain and a heavy chain, wherein thelight chain comprises a light chain variable region comprising an aminoacid sequence having at least 90%, or at least 91%, or at least 92%, orat least 93%, or at least 94%, or at least 95%, or at least 96% or atleast 97%, or at least 98% or at least 99% sequence identity with SEQ IDNO: 1 and the heavy chain comprises a variable heavy chain region and anFc region, wherein the heavy chain variable region comprises an aminoacid sequence having at least 90%, or at least 91%, or at least 92%, orat least 93%, or at least 94%, or at least 95%, or at least 96%, or atleast 97%, or at least 98%, or at least 99% sequence identity with SEQID NO:2 and the Fc region comprises an amino acid sequence having atleast at least 90%, or at least 91%, or at least 92%, or at least 93%,or at least 94%, or at least 95%, or at least 96%, or at least 97%, orat least 98%, or at least 99% sequence identity with SEQ ID NO: 3wherein the Fc region comprises one or a combination of substitutionsselected from the group consisting of C11S, C14S, and P23S.

Another embodiment (embodiment B) is an isolated antibody according toembodiment A, wherein the Fc region further comprises the amino acidsubstitution C5S.

In variations of the embodiments A and B the antibody comprises a lightchain variable region that does not comprise any of the substitutionsT33W, S26D, and Q27E.

In other variations of embodiments A and B, the light chain variableregion comprises the substitution R28K.

In some variations of the embodiments of A and B, the CDRs of the heavyand light chain have the sequences listed in Table 2.

TABLE 2 CDR1 light chain ISCRASQRVSSSTYSYMH (SEQ ID NO: 15)CDR2 light chain YASNLES (SEQ ID NO: 16) CDR3 light chainQHSWEIPPT (SEQ ID NO: 17) CDR1 heavy chain SYYMY (SEQ ID NO: 18)CDR2 heavy chain EINPSNGDTNFNEKFKS (SEQ ID NO: 19) CDR3 heavy chainSDGRNDMDS (SEQ ID NO: 20)

In yet other variation of embodiments A and B, the light chain variableregion comprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ IDNO:15). In still other embodiments, the light chain variable regioncomprises the amino acid sequence ICRRASQRVSSSTYSYMH (SEQ ID NO:15) andone or both of the amino acid sequences YASNLES (SEQ ID NO:16) andQHSWEIPPT (SEQ ID NO:17).

In some variations of embodiments A and B, the light chain variableregion comprises the amino acid sequence of SEQ ID NO:1. In yet otherembodiments the light chain variable region consists of the amino acidof SEQ ID NO:1. In some embodiments, the light chain consistsessentially of the amino acid sequence of SEQ ID NO:7. In otherembodiments, the light chain consists of the amino acid sequence of SEQID NO:7. In still other embodiments, the light chain comprises the aminoacid sequence of SEQ ID NO:11. In yet other embodiments, the light chainconsists essentially of the amino acid sequence of SEQ ID NO:11. Instill other embodiments, the light chain consists of the amino acidsequence of SEQ ID NO:11.

In other variations of the embodiments A and B, the antibody comprises aheavy chain variable region that does not comprise any of thesubstitutions T30H, Y33W, or S54N. In some embodiments of the antibodiesof embodiments A and B, the light chain variable region does notcomprise any of the substitutions T33W, S26D, and Q27E. In othervariations of embodiments A and B, the light chain variable region doesnot comprise any of the substitutions T33W, S26D, and Q27E and the heavychain variable region does not comprise any of the substitutions T30H,Y33W, or S54N.

In yet other variations of the embodiments A and B, the heavy chainvariable region comprises the substitution K74R. In one embodiment theheavy chain variable region comprises one or any combination of theamino acid sequences SYYMY (SEQ ID NO:18), EINPSNGDTNFNEKFKS (SEQ IDNO:19), and SDGRNDMDS (SEQ ID NO:20).

In another embodiment, the heavy chain variable region comprises theamino acid sequence of SEQ ID NO:2. In yet another embodiment the heavychain variable region consists essentially of the amino acid sequence ofSEQ ID NO:2. In still another embodiment the heavy chain variable regionconsists of the amino acid sequence of SEQ ID NO:2. In some embodiments,the heavy chain variable region comprises the amino acid sequence of SEQID NO:6. In yet other embodiments the heavy chain variable regionconsists essentially of the amino acid sequence of SEQ ID NO:6. In stillother embodiments the heavy chain variable region consists of the aminoacid sequence of SEQ ID NO:6.

One embodiment of the present invention is an isolated antibody, whereinthe light chain comprises the amino acid sequence of SEQ ID NO:1 and theheavy chain consists of the amino acid sequence of SEQ ID NO:9.

Another embodiment of the present invention is an isolated antibody,wherein the light chain consists of the amino acid sequence of SEQ IDNO:7 and the heavy chain consists of the amino acid sequence of SEQ IDNO:9.

Yet another embodiment is an isolated antibody wherein the light chainvariable region comprises the amino acid sequence of SEQ ID NO:5 and theheavy chain consists of the amino acid sequence of SEQ ID NO:9.

Still another embodiment is an isolated antibody wherein the light chainconsists of the amino acid sequence of SEQ ID NO:11 and the heavy chainconsists of the amino acid sequence of SEQ ID NO:9.

Yet another embodiment, is an isolated antibody wherein the light chainconsists of the amino acid sequence of SEQ ID NO:7 and the heavy chainconsists of the amino acid sequence of SEQ ID NO:13.

Another embodiment is an isolated antibody wherein the light chainconsists of the amino acid sequence of SEQ ID NO:11 and the heavy chainconsists of the amino acid sequence of SEQ ID NO:13.

In preferred embodiments, the antibody of the present invention isstable at 37° C. for a period of at least 12 hours.

In another aspect, the present disclosure provides methods for treatingsubjects having a CD40L-associated disease or disorder comprisingadministering to the subject a therapeutically effective amount of anantibody of the present invention. It is contemplated that an antibodyof the invention, or mixtures thereof, can be administered to thesubject as a monotherapy, which, as used herein, means that the antibodyis the only therapeutic agent administered to the patient that isdirected to the treatment of the underlying disease or disorder.Monotherapy using an antibody of the invention does not preclude theadministration of other drugs, non-limiting examples of which are musclerelaxants, nonsteroidal anti-inflammatory drugs, pain medications, andantidepressants. Accordingly, in various embodiments of the invention,one or a mixture of the antibodies of the invention, is the soletherapeutic agent directed to treatment of the underlying disease ordisorder.

It is also contemplated that the antibodies of the invention, ormixtures thereof, can be administered in combination with othertherapeutic agents. “In combination with” includes, but is not limitedto, administration of the therapeutic agents at different times, atdifferent frequencies, simultaneously, or combined in a single dosageform.

One embodiment is a method for treating a subject with aneurodegenerative or neuromuscular disease or disorder comprisingadministering to the subject a therapeutically effective amount of anantibody of the present invention. Neurodegenerative or neuromusculardiseases and disorders include, but are not limited to, Alzheimer'sDisease, Parkinson's Disease, Amyotrophic Lateral Sclerosis, MultifocalMotor Neuropathy, Primary Lateral Sclerosis, Spinal Muscular Atrophy,Kennedy's Disease, and Spinocerebellar Ataxia.

Another embodiment is a method for treating a subject with AmyotrophicLateral Sclerosis comprising administering to the subject atherapeutically effective amount of an antibody of the presentinvention.

One embodiment of the present invention is a method for treating asubject with an inflammatory or immune disease or disorder comprisingadministering to the subject a therapeutically effective amount of anantibody of the present invention. Inflammatory or immune diseases anddisorders include, but are not limited to, colitis, drug induced lupusnephritis, graft versus host disease, transplant rejection andatherosclerosis.

Still another embodiment is a method for treating a subject having anautoimmune disease comprising administering to the subject atherapeutically effective amount of an antibody of the presentinvention. Autoimmune diseases include, but are not limited to systemiclupus erythematous, type-1 diabetes, myasthenia gravis, inflammatorybowel disease, immune thrombocytopenic purpura and rheumatoid arthritis.

Yet another embodiment is method of inhibiting an immune response in asubject comprising administering to the subject a therapeuticallyeffective amount of an antibody of the present invention. In oneembodiment the immune response is graft vs. host disease. In anotherembodiment the immune response is organ transplant rejection.

In some embodiments, an antibody of the present invention isadministered as a monotherapy. In one embodiment the antibody is JB5 isadministered as monotherapy. In another embodiment the antibody JB5-K74Ris administered as monotherapy. In yet another embodiment the antibodyJB5-R28K is administered as monotherapy. In still another embodiment theantibody JB5-R28K-K74R is administered as monotherapy.

In some embodiments of the methods according to the present invention,the antibody is administered in combination with another therapeuticagent.

In some embodiments, the antibody of the present invention isadministered in combination with a compound that blocks the interactionbetween CD28 and CD86 or between CD28 and CD80.

In some embodiments the compound that blocks the interaction betweenCD28 and CD86 or between CD28 and CD80 is a CTLA4-Ig fusion protein. Inone embodiment the compound that blocks the interaction between CD28 andCD86 or between CD28 and CD80 is abatacept or belatacept or galiximab.

Pharmaceutical Compositions and Methods of Administration

To treat any of the foregoing disorders, pharmaceutical compositions foruse in accordance with the methods of the present disclosure may beformulated in a conventional manner using one or more physiologicallyacceptable carriers. Pharmaceutically acceptable carriers are determinedin part by the particular composition being administered, as well as bythe particular method used to administer the composition. Accordingly,there are a wide variety of suitable formulations of the compoundsuseful in the methods of the present disclosure (see, e.g., Remington:The Science and Practice of Pharmacy, 20th ed., Gennaro et al. Eds.,Lippincott Williams and Wilkins, 2000).

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

According to the present disclosure the compounds can be administered byany suitable means, which can vary, depending on the type of disorderbeing treated and on the nature of the compound itself. For example, forthe antibodies of the present invention, administration routespreferably include parenteral, e.g., intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous. Preferably, theparenteral dosing is given by injection, most preferably intravenous,intramuscular or subcutaneous injection. The amount to be administeredwill depend on a variety of factors such as the clinical symptoms,weight of the individual, and whether other drugs are administered. Itshould be appreciated that determination of proper dosage forms, dosageamounts, and routes of administration is within the level of ordinaryskill in the pharmaceutical and medical arts.

EXAMPLES

The following examples illustrate the methods used to make and test theantibodies of the invention. Suitable modifications and adaptations ofthe described conditions and parameters normally encountered in the artof molecular biology and immunology will be apparent to one of skill inthe art.

Example 1: Antibody Production

In order to produce the antibodies of the invention, nucleic acidsequences encoding the heavy chain and the light chain of the desiredantibody were designed to be suitable for expression in mammalian cellssuch as Chinese Hamster Ovary (CHO) cells. The nucleic acids were thenartificially synthesized and ligated into the antibody expression vectorBPJPuro using standard molecular biology techniques. BPJPuro is a dualgene mammalian expression vector optimized for selectable and stableexpression of immunoglobulins in Chinese Hamster Ovary (CHO) cells. Thevector is then transfected into CHO cells and stable transfectantsselected.

Production of JB5 Antibodies

A nucleic acid (SEQ ID NO:10) encoding a heavy chain having the aminoacid sequence of SEQ ID NO:9, and a nucleic acid (SEQ ID NO:8) encodinga light chain having the amino acid sequence of SEQ ID NO:7, weresynthesized and ligated into the antibody expression vector BPJPuro.

The resulting expression vector encoding the heavy and light chains wastransfected into the CHO line (CHO SA, Cellectis SA, Paris, France)using liposome mediated transfection. Stable transfectants were isolatedby puromycin selection and subcloned to provide clonal cell lines.Candidate cell lines were adapted to serum free suspension culture andscreened for IgG production and robust growth. One of the cell lines wasselected and named JB5, the cell line was cultured in a pilot scalebioreactor and the antibody JB5 was purified from conditioned medium bysequential concentration, Protein A/G affinity chromatography, and sizeexclusion chromatography.

Example 2: CD40L Binding Assay

A three part sandwich ELISA assay was used to determine binding kineticsof the JB5 antibody relative to the parental antibody hu5c8. All washeswere performed using 3 washes of 250 μl of PBS. A 96-well polystyreneplate was coated with 100 μl/well of JB5 or hu5c8 antibody (2 μg/ml) for16 hours at 4° C. The plate was washed and then blocked with 2% bovineserum albumin/PBS for 1 hour at room temperature. The plate was washedand recombinant human CD40L protein (Santa Cruz Biotechnology, SantaCruz, Calif., USA) was added to the plate titrated out by 2-folddilution starting at 2000 ng/ml. After binding and washing, the boundCD40L protein was detected using 100 μl a biotinylated goat anti-humanCD40L polyclonal antibody (200 ng/ml) and 100 μl astreptavidin-horseradish peroxidase conjugate at 100 ng/ml. Colorimetricdetection was performed with the chromagen TMB(3,3′,5,5′-tetramethylbenzidine) and spectrophotometric analysis ofabsorption at 450 nm. The resulting binding curves (FIG. 3) show thatJB5 (circle) has highly similar CD40L binding relative to the parentalantibody hu5c8 (square). The control protein CTLA4-IgG1 (triangle),having the same Fc domain as JB5 showed no significant binding. Thecalculated EC50 for hu5c8 and JB5 is 114 and 137 nM, respectively.JB5-R28K and JB5-K74R showed binding similar to that of JB5.

Example 3: Fc Gamma Receptor Binding Assays

hu5c8/Human Fc Gamma Receptor Binding Assay

A solid phase ELISA binding assay was performed to determine the levelof binding of four human Fc gamma receptor isoforms to the parentalhu5c8 antibody. 100 μl/well hu5c8 antibody (2 μg/ml in phosphatebuffered saline) was added to the wells of a 96 well polystyrene plateand incubated for 16 hours at 4° C. The plate was blocked andrecombinant human Fc gamma receptor (FCGR) proteins (Santa CruzBiotechnology, Santa Cruz, Calif.) titrated by 2-fold dilution with astarting concentration of 5 μg/ml. Four recombinant FCGR isoforms weretested separately as follows: FCGR1A (CD64) (SEQ ID NO:22), FCGR2A(CD32) (SEQ ID NO:23), FCGR3A (CD16a) (SEQ ID NO:24), FCGR3B (CD16b)(SEQ ID NO:25). After binding and washing, the FCGR was detected usingan appropriate FCGR isoform specific murine monoclonal antibody (1000ng/ml) and a horseradish peroxidase conjugate goat anti-mouse IgGdetector antibody. Colorimetric detection was performed with thechromagen TMB (3,3′,5,5′-tetramethylbenzidine) and spectrophotometricanalysis of absorption at 450 nm. The resulting binding curves (FIG. 4)demonstrate that the parental hu5c8 antibody binds the high affinityFCGR1A (circle, solid line) receptor (SEQ ID NO:22) and the FCGR2Areceptor (SEQ ID NO:23) (circle, dotted line) expressed on activatedplatelets, with high affinity. The hu5c8 antibody showed no binding tothe FCGR3A receptor (SEQ ID NO:24) or FCGR3B receptor (SEQ ID NO:25)isoforms.

JB5-Human Fc Gamma Receptor Binding Assay

A solid phase binding assay was used to test binding of human Fc gammareceptor isoforms to the mutant JB5 antibody. 100 μl/well JB5 (2 μg/mlin phosphate buffered saline) was coated for 16 hours onto a 96 wellpolystyrene plate. The plate was blocked and recombinant human Fc gammareceptor (FCGR) proteins (Santa Cruz Biotechnology, Santa Cruz, Calif.)titrated onto by 2-fold dilution with a starting concentration of 5μg/ml. Four recombinant FCGR isoforms were tested separately as follows:FCGR1A (CD64) (SEQ ID NO:22), FCGR2A (CD32) (SEQ ID NO:23), FCGR3A(CD16a) (SEQ ID NO:24), FCGR3B (CD16b) (SEQ ID NO:25). After binding andwashing the FCGR was detected using an appropriate FCGR isoform specificmurine monoclonal antibody (1000 ng/ml) and a horseradish peroxidaseconjugate goat anti-mouse IgG detector antibody. Colorimetric detectionwas performed with the chromagen TMB (3,3′,5,5′-tetramethylbenzidine)and spectrophotometric analysis of absorption at 450 nm. The resultingbinding curves (FIG. 5) demonstrate that the JB5 antibody binds neitherthe high affinity FCGR1A receptor (SEQ ID NO:22) nor the FCGR2A receptor(SEQ ID NO:23), expressed on activated platelets, in this assay. Likethe parental hu5c8 antibody, no binding was observed for FCGR3A receptor(SEQ ID NO:24) or FCGR3B receptor (SEQ ID NO:25).

Example 4: Stability of JB5 at 22° C. and at 37° C.

Because JB5 lacks three of the disulfide linkages in wild-type IgG1antibodies, JB5 was tested using size exclusion chromatography todetermine if the antibody was stable, i.e., existed as a tetrameric,fully intact antibody. Hu5c8, which has the three disulfide linkages wasused as a control.

Two experiments were performed, each comparing JB5 with hu5c8. In thefirst experiment, the antibodies were at room temperature (22° C.)before and during chromatography. To simulate in vivo conditions, in thesecond experiment the antibodies were incubated in human plasma at 37°C. for 30 minutes prior to chromatography at 30° C. Twenty micrograms ofJB5 or hu5c8 in PBS was injected into a TSK® gel G3000SW (7.8 mm×30 cm,5 μm bead column) equipped with a pre-column filter TSK® gel Guard SWxl, (6.0 mm×4.0 cm, 7 μm bead column) (Tosoh Bioscience, King ofPrussia, Pa.). The mobile phase was PBS and the elution rate was 1.0mL/minute and the absorbance was measured at 280 nm. At both 22° C. andat 30° C. JB5 had an observed molecular weight of 183 kDa (FIG. 6) andhu5c8 (FIG. 7) had a MW of 164 kDa consistent with the antibody being inthe tetrameric, divalent form. The observed 19 kDa difference betweenthe hu5c8 antibody and JB5 may be due to increased glycosylation of theFc domain of JB5.

Example 5: Elimination of Platelet Activation

In order to determine the effect of JB5 on CD40L immune complex mediatedplatelet activation, the antibody was assayed for its ability to inducethe platelet cell surface marker protein PAC-1. Whole blood was drawnfrom three healthy volunteers into 3.2% Na citrate tubes discarding thefirst 2 ml. Platelet rich plasma was prepared by centrifugation for 15minutes at 120 g the platelet count was normalized with phosphatebuffered saline to 1×10⁵ cells/ml. Immune complexes of recombinant humanCD40L (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and the testantibodies, hu5c8, JB5, and hu5c8 F(ab′)₂ were prepared at aCD40L:Antibody molar ratio of 3:1 (0.6944 nmole CD40L:0.2315 nmoleantibody) by preincubation at room temperature for 15 minutes. Theimmune complex mixture was diluted to a final concentration of 5 μg/mlCD40L in the normalized PBS/platelet solution and incubated at 37° C.for 30 minutes. Negative controls were untreated platelets and CD40Lalone. The platelet activation positive control was prepared by theaddition of ADP to a final concentration of 20 micromolar in thenormalized PBS-platelet solution. After 30 minutes of incubation,anti-human PAC-1-FITC conjugated antibody was added to all samples andincubated for 15 minutes. Samples were diluted 1:1 into 2%paraformaldehyde:PBS buffer, fixed on ice for 30 minutes, centrifuged at100 g, for 5 minutes to pellet the cells. The cells were resuspended inPBS. Fluorescence activated cell sorting (FACS) was performed on a GuavaeasyCyte flow cytometer (EMD Millipore, Inc., Billerica, Mass., USA).Post-acquisition analysis was performed using FlowJo software (FlowJo,LLC, Ashland, Oreg., USA).

An untreated platelet control sample was used to set negative andpositive PAC-1 activation gates (FIG. 8). Platelets activated with 20micromolar ADP had a significant increase in PAC-1 cell surfaceexpression (FIG. 9). Consistent with published observations, see e.g.,Mirabet, M., et al., Molecular Immunology 45, 937-944 (2008), CD40Lalone was able to activate platelets at a low level (FIG. 10). Thisactivation was significantly increased when CD40L was present with hu5c8antibody as an immune complex (FIG. 11). In contrast, the engineeredantibody JB5 complexed with CD40L demonstrated very low levels ofplatelet activation (FIG. 12). This reduction in the activationpotential of a CD40L:JB5 immune complex is mediated by the loss of FcRinteraction because the hu5c8 F(ab′)2:CD40L immune complex (FIG. 13)also did not activate platelets relative to the hu5c8-IgG1:CD40L immunecomplex (FIG. 11). FIG. 14 shows the platelet activation results fromthree persons' platelets after incubation of the platelets with 20 μMADP, 5 μg/ml CD40L, the immune complex of CD40L and hu5c8, the immunecomplex of CD40L and JB5 antibody or the immune complex of CD40L withhu5c8 F(ab′)₂. The JB5 immune complex showed no significant plateletactivation when compared to the immune complex of CD40L with hu5c8F(ab′)₂ platelets (p<0.34 (Unpaired T test, 2 tailed; t=1.013, df=4).Further, the JB5 immune complex showed significantly less plateletactivation when compared with the hu5c8 immune complex (p<0.005(Unpaired T test, 2 tailed; t=5.586, df=4).

While a number of embodiments of this disclosure are described, it isapparent that the basic examples may be altered by one skilled in theart to provide other embodiments that use or encompass methods andprocesses of this invention. The embodiments and examples are forillustrative purposes and are not to be interpreted as limiting thedisclosure, but rather, the appended claims define the scope of thisinvention.

What is claimed is:
 1. An isolated antibody that binds to CD40L and thatcomprises a light chain and a heavy chain, wherein (i) the light chaincomprises a light chain variable region comprising an amino acidsequence having at least 98% sequence identity with SEQ ID NO:1; (ii)the heavy chain comprises a heavy chain variable region and an Fc regionwherein a) the heavy chain variable region comprises an amino acidsequence having at least 98% sequence identity with SEQ ID NO:2; and b)the Fc region comprises an amino acid sequence having at least 98%sequence identity with SEQ ID NO:3 wherein the Fc region comprises oneor a combination of amino acid substitutions selected from the groupconsisting of C11S, C14S, and P23S.
 2. The isolated antibody accordingto claim 1, wherein the Fc region comprises the amino acid substitutionsC11S, C14S, P23S and further comprises the amino acid substitution C5S.3. The isolated antibody according to claim 1, wherein the light chainvariable region does not comprise the substitution T33W, S26D, or Q27E.4. The isolated antibody according to claim 1, wherein the light chainvariable region comprises the substitution R28K.
 5. The isolatedantibody according to claim 2, wherein the light chain variable regioncomprises the amino acid sequences ISCRASQRVSSSTYSYMH (SEQ ID NO:15),YASNLES (SEQ ID NO:16), and QHSWEIPPT (SEQ ID NO:17).
 6. The isolatedantibody according to claim 1, wherein the heavy chain variable regiondoes not comprise the substitution T30H, Y33W, or S54N.
 7. The isolatedantibody according to claim 1, wherein the heavy chain variable regioncomprises the substitution K74R.
 8. The isolated antibody according toclaim 5, wherein the heavy chain variable region comprises the aminoacid sequences SYYMY (SEQ ID NO:18), EINPSNGDTNFNEKFKS (SEQ ID NO:19),and SDGRNDMDS (SEQ ID NO:20).
 9. The isolated antibody according toclaim 1, wherein the Fc region comprises the amino acid sequence of SEQID NO:4; and (i) the light chain variable region comprises the aminoacid sequence of SEQ ID NO:1; and the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO:2; or (ii) the lightchain variable region comprises the amino acid sequence of SEQ ID NO:1;and the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:6; or (iii) the light chain variable region comprises theamino acid sequence of SEQ ID NO:5; and the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO:2; or (iv) the lightchain variable region comprises the amino acid sequence of SEQ ID NO:5;and the heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:
 6. 10. The isolated antibody according to claim 1, wherein(i) the light chain consists of the amino acid sequence of SEQ ID NO:7;and the heavy chain consists of the amino acid sequence of SEQ ID NO:9;or (ii) the light chain consists of the amino acid sequence of SEQ IDNO:7; and the heavy chain consists of the amino acid sequence of SEQ IDNO:13; or (iii) the light chain consists of the amino acid sequence ofSEQ ID NO:11; and the heavy chain consists of the amino acid sequence ofSEQ ID NO:9; or (iv) the light chain consists of the amino acid sequenceof SEQ ID NO:11; and the heavy chain consists of the amino acid sequenceof SEQ ID NO:13.
 11. A method for treating a subject with a CD40Lassociated disease or disorder comprising administering to the subject atherapeutically effective amount of an isolated antibody wherein theantibody binds to CD40L and comprises a light and a heavy chain, wherein(i) the light chain comprises a light chain variable region comprisingan amino acid sequence having at least 98% sequence identity with SEQ IDNO:1; (ii) the heavy chain comprises a heavy chain variable region andan Fc region wherein a) the heavy chain variable region comprises anamino acid sequence having at least 98% sequence identity with SEQ IDNO:2; and b) the Fc region comprises an amino acid sequence having atleast 98% sequence identity with SEQ ID NO:3 wherein the Fc regioncomprises wherein the Fc region comprises the amino acid substitutionsC11S, C14S, P23S and C5 S; wherein the disease or disorder is aneurodegenerative or neuromuscular disease or disorder; an inflammatoryor immune disease or disorder; or an autoimmune disease.
 12. The methodaccording to claim 11, wherein the antibody is administered incombination with another therapeutic agent.
 13. The method according toclaim 12, wherein the antibody is administered in combination with acompound that blocks the interaction between CD28 and CD86 or betweenCD28 and CD80.
 14. The method according to claim 13, wherein thecompound that blocks the interaction between CD28 and CD86 or betweenCD28 and CD80 is a CTLA4-Ig fusion protein.
 15. The method according toclaim 14, wherein the compound that blocks the interaction between CD28and CD86 or between CD28 and CD80 is abatacept or belatacept orgaliximab.
 16. The method according to claim 11, wherein the disease ordisorder is a neurodegenerative disorder or a neuromuscular disorderselected from the group consisting of Alzheimer's Disease, Parkinson'sDisease, Amyotrophic Lateral Sclerosis, Multifocal Motor Neuropathy,Primary Lateral Sclerosis, Spinal Muscular Atrophy, Kennedy's Disease,and Spinocerebellar Ataxia.
 17. The method according to claim 11,wherein the disease or disorder is an inflammatory or immune disease ordisorder selected from the group consisting of colitis, drug inducedlupus nephritis, graft versus host disease, transplant rejection andatherosclerosis.
 18. The method according to claim 11, wherein thedisease or disorder is an autoimmune disease, selected from the groupconsisting of systemic lupus erythematous, type-1 diabetes, myastheniagravis, inflammatory bowel disease, immune thrombocytopenic purpura andrheumatoid arthritis.
 19. A method of inhibiting an immune response in asubject comprising administering to the subject a therapeuticallyeffective amount of an isolated antibody wherein the antibody binds toCD40L and comprises a light and a heavy chain, wherein (i) the lightchain comprises a light chain variable region comprising an amino acidsequence having at least 98% sequence identity with SEQ ID NO:1; (ii)the heavy chain comprises a heavy chain variable region and an Fc regionwherein a) the heavy chain variable region comprises an amino acidsequence having at least 98% sequence identity with SEQ ID NO:2; and b)the Fc region comprises an amino acid sequence having at least 98%sequence identity with SEQ ID NO:3 and wherein the Fc region comprisesthe amino acid substitutions C11S, C14S, P23S and C5S.
 20. The method ofclaim 19, wherein the immune response is graft vs. host disease or organtransplant rejection.